The viable cell enumeration, or count, is the number of viable cells per volume of sample, and is denoted here by .rho..sub.s. The determination of a viable enumeration is important to and widely used in biological research, clincial microbiology, cancer diagnosis and treatment, environmental science, food safety, toxicology, and research and development in basic and applied biology. Present methods for viable enumeration are slow and generally labor intensive, and many newer methods which purport to give an enumeration are not based on actual viable cell counts. Instead, many of these methods measure some average property of a large number of cells which, under well defined conditions, correlates with a count, but which under other conditions generally does not correlate accurately with a viable count.
Present cell analysis methods involve two major classes of assays. The first class rapidly detects and identifies specific cells directly from a primary sample, but does not determine cell viability. The most widely used in this class are specific ligand binding assays, e.g. immunoassays and genetic probes. However, they require many cells, and do not distinguish between dead and viable cells. This restricts their use to samples in which sufficient numbers of cells are present, and to determinations in which direct assessment of the physiological state of the cell is irrelevant. The second class of assays is used for viable cell determinations either directly using the primary sample, or using a subculture of the primary sample. The most traditional and widely used method is the plate count, which allows determination of single cell viability, based on growth, under many test conditions (see, for example, Hattori The Viable Count: Quantitative and Environmental Aspects, Brock/Springer, Madison, 1988). An important attribute of viable plate enumeration is that the time required to obtain a determination is independent of the concentration of the cell in the sample, as formation of each colony proceeds from an initial single cell. The major disadvantage is its slowness, as typical determinations require one-half to several days, and are also labor- and materials-intensive.
The disadvantages of viable plating can better be appreciated by drawing attention to its basic attributes. Viable plating is a well established, important method for qualitatively determining the growth of cells, particularly the presence or absence of growth for given conditions, and is often based on the growth of initial cells into distinct colonies. Viable plating typically involves the spreading of a suspension of cells onto the surface of a gel-containing petri dish, with or without the pouring of a gel layer over the first gel surface. The gels are provided with nutrients, such that following an incubation period at a suitable temperature, many generations of growth occur, which leads to formation of visible colonies. For many microorganisms formation of visible colonies requires growth for 22 to 30 generations and therefore colonies producing containing 10.sup.7 to 10.sup.9 cells. (See Sharpe, in Mechanizing Microbiology, A. N. Sharpe and D. S. Clark (Eds.) Charles C. Thomas, Springfield, 19-40, 1978). Although conventional viable plating leads to formation of colonies, and thereby provides a basis for counting viable cells by counting colonies, the presence or absence of colonies only allows an inference that the conditions present in the gel support do or do not support growth. For this reason, conventional viable plating is not well suited to quantitative determinations such as cell growth rate and lag time, because viable plating based on visual inspection counts the number of colonies formed, but does not determine how the cellular material or amount of cellular constituents in the colonies varies with time. An additional complication arises because the nutrient and metabolite concentrations within a colony comprise a microenvironment, which generally changes with time in an variable way as microcolonies increase to form larger colonies with many cells in close proximity. The microenvironment within a large colony can also have significant heterogeneity of chemical composition within the microcolony, so that different cells within a large colony experience different growth conditions. Further, although some methods are based on a straightforward extension and application of scanning optical methods for determination of optical properties of colonies on or in gel slabs, such methods suffer from relatively large cost and size, and, because of the relatively large gel slab size, do not allow incubation conditions to be changed rapidly at the site of the cells within the gel. (See Glaser in New Approaches to the Identification of Microorganisms Proceedings of a Symposium on Rapid Methods and Automation in Microbiology, C. -G. Heden and T. Illeni (Eds.), Wiley, N.Y., 3-12, 1975).
Instrumented methods for rapidly determining cell or culture growth and/or metabolic activity have been developed which only partially address the limitations of the viable plate assay. These include optical techniques for growth determination such as those which measure the change in light scattering due to many cells in a liquid suspended culture (See, for example, Edberg and Berger, in Rapid Methods and Automation in Microbiology and Immunology, K. O. Habermehl, Ed., Springer-Verlag, Berlin, 215-221, 1985), and a variety of metabolic activity based techniques which measure changes due to many cells in an analyzed sample. Examples include changes in extracellular pH (See, for example, Cowan and Steel's Manual for the Identification of Medical Bacteria, Cambridge University Press, Cambridge, 1974; Manual of Methods for General Bacteriology, P. Gerhardt, (Ed), American Society for Microbiology, Washington, 1981), carbon dioxide release (see, for example, Courcol et al., J. Clin. Microbiol., 25: 26-29, 1986; Manca et al., J. Clin. Microbiol., 23: 401-403, 1986), electrical impedance (see, for example, Stewart, J. Exp. Med., 4: 235-245, 1899; Eden and Eden Impedance Microbiology, Research Studies Press, Letchworth, 1984; Hadley and Yajko, in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 193-209, 1985; Bishop and White, J. Food Protect., 49: 739-753, 1986), chemiluminescence (see, for example, Neufeld et al., in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 51-65, 1985; Rapid Methods and Automation in Microbiology and Immunology, Habermehl (Ed.), Springer-Verlag, Berlin, 1985) or fluorescence (see, for example, Rossi and Warner in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 1-50, 1985; Rapid Methods and Automation in Microbiology and Immunology, Habermehl (Ed.), Springer-Verlag, Berlin, 1985). A disadvantage of all such metabolic activity methods is that they are based on combined effects of a large number of cells, and therefore generally require an initial process, based on plating, to obtain initial colonies for purposes of inoculation of the analyzed sample, such that the determinations based on many cells at least are based on a monopopulation, i.e. a population comprised nominally of the same type of cells. For this reason, although a total population cell determination may itself be rapid, it is generally preceeded by a viable plating method, or its equivalent, which is slow. Thus, the total analysis time, counted from receipt of a primary or non-plated sample to a cell growth determination, is the sum of both, and therefore still long.
Further, because such determinations are based on the combined effect of a large, but unkown number, of cells, such total population determinations do not actually yield a count. In contrast, determinations based on many individual measurements, each associated with an initial single cell, can yield a count.
Finally, because these total population methods are based on the combined effects of many cells, the time required for a determination becomes significantly longer as the number of cells decreases, i.e. as the sample's cell concentration decreases.
Similarly, prior use of flow cytometry for cell growth measurements (see, for example, Hadley et al. in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 67-89, 1985) is limited, because conventional use of flow cytometry performs measurements on individual cells, or clumps of cells which naturally adhere, in an aqueous liquid suspension, and therefore does not have the capability to measure colony formation. For this reason, prior use of flow cytometry can only measure total numbers of cells in a volume in order to determine average growth, and must also, therefore, involve a careful volume measurement, and is dependent on the signal-to-noise ratio of single cell measurements. This signal-to-noise ratio is less than satisfactory for many measurements (see, for example, Shapiro Practical Flow Cytometry, R. Liss, New York, 1985; Hadley et al. in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 67-89, 1985).
Likewise, quantitative microscopy and image analysis combined with conventional gel preparations, such as gel slabs, petri dishes and the like, although capable of determining colony formation, is tedious in manual versions, and conventional gel slabs, petri dishes and the like cannot provide physical manipulability or a sufficiently fast (small) characteristic diffusion time within the gel, so that cells cannot be rapidly and conveniently exposed to different growth conditions, such as rapid changes in concentrations of nutrients, drugs, hormones, enzymes, antibodies and other chemicals. In addition, conventional gel slabs, petri dishes and the like cannot be readily manipulated physically because of their size, and therefore cannot be readily used for exposure of gel-entrapped cells to in vivo conditions.
For example, agarose slabs can be used to protect fragile biological entities such as plant protoplasts in cases wherein both free cells and cells entrapped in agarose beads are found to be damaged (see Pasz and Lurquin, Bio Techniques 5: 716-718, 1987), but such gel slabs have relatively long characteristic diffusion times and cannot be readily inserted into animals in order to provide biological influence.
In another example, guiding human cancer chemotherapy by determining human cancer cell growth in immunodeficient mice, which are exposed to trial chemotherapy conditions, can be approached by insertion of a small tissue piece into a mouse (see Bogden, Annal. Chirurgiae Gynaecol., Vol. 74 Suppl. 199, 12-27, 1985; Favre et al., Eu. J. Can. Che. Oncol., 22: 1171-1178, 1986), but the insertion of conventional gel slabs or petri dishes into the mice would be similarly cumbersome, requiring considerable manual manipulation. For example, slices of clot about 1 to 1.4 mm on a side containing about 1.times.10.sup.5 to 2.times.10.sup.5 human cancer cells can be prepared and inserted into a mouse, and subsequent combined growth due to this large number of cells can be determined by measurement of the resulting tumor dimensions. Colony formation is not determined, however, so that such procedures both require manual manipulations and do not result in cell preparations which are readily analyzed quantitatively for cell growth, such as average cell growth rate, or more importantly, the quantitative growth of individual cells (see Fingert, Cancer Res., 47: 3824-3829, 1987). Further, in the absence of applied physical forces, transport of molecules within gel materials occurs by diffusion, for which the characteristic diffusion times are dependent on the square of the thickness of the gel slabs or petri dish gel material, which thicknesses are typically 10.sup.-1 to 3.times.10.sup.-1 cm or larger. For this reason characteristic diffusion times for even small molecules are long, as can be estimated by calculating t.sub.diffusion .apprxeq.x.sup.2 /D, where x is the characteristic dimension or thickness of the gel. The diffusion constant, D, is about 10.sup.-5 cm.sup.2 /sec for small molecules, and is significantly smaller, often about 10.sup.-7 cm.sup.2 /sec for large molecules such as antibodies and enzymes, so that typically t.sub.diffusion .apprxeq.17 to 50 minutes for small molecules, and about 2.8 to 8.3 hours for large molecules. It is generally well known that times of about 3t.sub.diffusion to 5t.sub.diffusion are required to obtain changes which are 95 to 99% complete for diffusion controlled processes. Thus, the times corresponding to 3t.sub.diffusion for a 95% complete change are about 50 minutes to about 21/2hours for small molecules, and about 81/2hours to about 1 day for large molecules, and still longer for a 99% complete change, so that it is generally impossible to provide rapid, completed changes of chemical concentrations at the site of cells contained in conventional gel slabs, Petri dishes or large gel particles by the general means of changing external chemical concentrations (see, for example, Nilsson et al., Nature, 302: 629-630, 1983; Nilsson et al., Eur. J. Appl. Microbiol. Biotechnol., 17: 319-326, 1983).
Similarly, prior methods for determining the effects of compounds and agents on the growth of viruses involve the use of cell culture, such that sample viruses infect one or more provided cells, and grow within the infected cells, often resulting in cell lysis and releasing a large number of viruses. Prior methods for determining growth of chlamydia also involve growth within host cells.
Further, prior methods for determining growth of nucleic acids involve a cyclic incubation which results in a doubling of specific nucleic acids with each cycle (see Mullis et al., U.S. Pat. No. 4,683,195). Finally, prior methods for determining growth of immunological complexes such as antibody-antigen complexes are carried out in a homogeneous phase wherein the reactants are in solution, or using a solid phase, wherein one or more reactants are attached to a solid surface.
The optical path length associated with optical measurements on cells in conventional gel slabs or Petri dishes can often be equal to a significant fraction of the thickness, or the entire thickness, of the gel slabs or petri dish material. For this reason, the size of conventional, macroscopic gel preparations also places limitations on optical measurement methods which can be used with the gel preparations, as light absorbtion, light scattering and auto fluorescence can be significant, and result in difficulty in making accurate measurements on individual or small numbers of cells within such macroscopic gels.
A major disadvantage is the slowness of viable plate enumerations: typical determinations require one-half to several days, and are also labor- and materials-intensive. Another major disadvantage is the relatively inability to readily change the chemical or physical conditions present during incubation, as the macroscopic size of petri dishes results in slow diffusion times for changes of chemical conditions, slow temperature response times, and a general inability to position the cells on the surface of a petri dish in close proximety of conditions, or sources of influence, which can alther cell survival, cell growth or other cell behavior.
In addition, prior use of microdroplets (MDs), although indicating that information relating to the viable enumeration can be estimated, is highly approximate, generally utilizes only one or a small number of MD sizes, is dependent upon interpretation of visual images of MDs while observing MDs by microscopy, and suffers from being tedious and difficult. For example, the investigation by Rotman of the activity of individual moleucles of the enzyme .beta.-D-galactosidase depended upon the visual inspection of a relatively small number of MDs by fluorescence microcospy (PNAS, 47: 1981-1991, 1961). Further, although this investigation of individual enzyme activity is important and fundamental, the methodology is sufficiently tedious and difficult that this use of MDs has not been repeated or extended, and has not been available in a more automated or more quantitatively accurate version. A related investigation of bacterial .beta.-D-Galactosidase activity utilized bacteria contained within liquid microdroplets (Revel et al, PNAS 47: 1956-1967, 1961).
Likewise, prior use of gel microdroplets (GMDs) has involved the detection of microorganisms in GMDs surrounded by mineral oil, with extracellular light absorbance or fluorescence changes manually observed by microscopy, with manual estimates of GMD diameter by comparision to a haemocytometer grid dimension, such that a very approximate estimate of the GMD volumes within one or a small number of GMD size ranges could be made (Weaver et al., Ann. N.Y. Acad. Sci., 434: 363-372, 1984; Williams et al, Ann. N.Y. Acad. Sci. 501: 350-353, 1987). Because of the tedious, manual nature of such determinations, typically the diameters of 100 or fewer GMDs were estimated, and used to approximately estimate the consistency of the observed number of occupied GMDs with Poisson statistics.
Although approximate consistancy with Poisson statistical formulae was found in prior use of liquid and gel microdroplets, such prior use of MDs suffered from inaccuracy of MD size measurement, an inability to readily measure large numbers of MDs, and a resultant inability to apply mathematical statistical analysis in a more thorough, iterative fashion that would provide self-consistent mathematical determinations of the occupation of the MDs, and, thereby, an enumeration. Further, such prior use of MDs did not provide a stringent basis for determining the viability of any biological entities contained within MDs, and therefore did not provide the type of measurent yielded by conventional viable plating methods.
Further, prior use of liquid microdroplets (LMDs) has been limited to demonstrating that the occupation of LMDs by single enzyme molecules (Rotman, PNAS 47: 1981-1991, 1961) or by bacteria (Revel et al, PNAS 47: 1956-1967, 1961), and of GMDs by bacteria and yeast, is consistent with Poisson statistics (Weaver et al., Ann. N.Y. Acad. Sci., 434: 363-372, 1984; Williams et al, Ann. N.Y. Acad. Sci. 501: 350-353, 1987). However, such prior demonstrations do not have high accuracy, as the measurements of such LMDs and GMDs surrounded by a non-aqueous fluid are based on visual observation of a relatively small number of MDs using light microscopy or fluorescence microscopy, and do not involve a large number of measurements of individual MD diameters, from which MD volume for each adherent, somewhat deformed spherical MD can be estimated within each of several ranges of MD volumes. For this reason, instead of being directed towards a determination of an enumeration, these prior demonstrations emphasized approximate consistancy with Poisson statistics. Further, these prior uses of MDs have not involved determination of the viability of cells based on growth. For these reasons, prior use of MDs has not been directed towards a rapid determination of a viable enumeration.
Thus, methods which provide more rapid viable enumeration of biological entities than conventional viable plating, which is based on viability determined directly by biological growth of biological entities, or determined less directly by vital stains, which is capable of measuring large numbers of biological entities, which is capable of more accurate quantitation, and which is capable of more automation, would be highly desirable.